One of the next great challenges of the postgenomic era is functional analysis of the proteome in space and time, which will be essential to understand normal and pathological cell behavior. Direct analysis of protein function in complex intracellular processes requires a method to acutely, rapidly and specifically inactivate proteins of interest in real time and in selective regions of live cells. Such a method does not exist. Current methods to investigate intracellular protein function have severe limitations, and are either non-specific or lack sufficient spatial and temporal resolution. For example, because RNA interference (RNAi) relies on slow intracellular protein turnover, it is useful to detect long-term phenotypes, but does not allow direct, acute analysis of protein function. Small molecule inhibitors are not broadly applicable because specificity is often hard to establish in live cell experiments, and it is challenging to design inhibitors of non-enzymatic protein functions. In addition, both of these methods can only be applied to whole cells and are not useful to analyze spatially restricted intracellular processes. Finally, photoablation and chromophore-assisted laser inactivation (CALI) employ non-specific, non-reversible protein destruction using high power illumination. The objective of this project is to address this challenge by developing an innovative, versatile, genetically-encoded method by which a protein of interest can be disrupted by specific light-activated proteolysis in either whole cells or intracellular regions as a novel tool to analyze protein function in live cells. Because we propose to use light to toggle protease activity, experiments can be carried out entirely on an adequately equipped microscope allowing unprecedented high temporal and spatial control of intracellular protein inactivation by using patterned illumination. Such a technique would revolutionize cell biology, and would have an exceptionally high impact on the analysis of intracellular processes that occur on short time scales, and rely on direct regulation of protein activity rather than gene expression changes. The strategy to achieve this objective will involve two major steps: 1) Design and optimize a light-activated site-specific protease by combining the photosensory domain of plant phototropins with the exceptionally high specificity of picornavirus 3C proteases; and 2) Validate feasibility by genetically engineering protease-sensitive proteins of interest, and analyze functional consequences of light-activated target protein inactivation in live cells in which endogenous function of the gene of interest has been silenced by RNAi. We will test our approach by generating protease-sensitive versions of two multi-domain cytoskeleton proteins, talin and EB1, and by constructing a protease-sensitive kinase domain to demonstrate feasibility and versatility. PUBLIC HEALTH RELEVANCE: This project aims to build a novel tool to inactivate specific proteins in live cells with high spatial and temporal control by developing a light-activated site-specific protease in combination with a protease-sensitive version of a target protein of interest. A method to specifically, rapidly and locally disrupt intracellular protein function does not currently exist, and development of such a tool will have a high impact on the analysis of intracellular protein function in many fields of biomedical research. Detailed analysis of protein function in live cells is required to understand normal and pathological processes in cells, and will lead to the development of novel drugs and therapeutic strategies.